In the manufacture of proteins or protein precursors the separation of glycosylated proteins from non-glycosylated proteins represents an independent field of research. The industrial implications of the present invention relate to the optimisation of protein purification. It is the purpose of the optimised purification to achieve an end product to be used commercially comprising non-glycosylated proteins, substantially free from glycosylated proteins.
Proteins or protein precursors may originate from yeast expression systems. A correlation between a high expression level and an increase in glycosylated protein precursors has been observed. As a consequence the need for a more efficient process of separating the non-glycosylated proteins from the glycosylated proteins has become even more apparent.
In the yeast expression system a yeast organism produces proteins or protein precursors synthesised intracellularly. The yeast host organism is transformed by an expression vehicle harbouring DNA encoding the desired protein. The process comprises preparing a culture of the transformed yeast host organism, growing the culture and recovering the protein from the culture medium.
Prior art recovery of the desired protein comprised multiple steps of purification using the process of chromatography, such as ion exchange chromatography involving an eluant containing salt. By using the method of reverse phase high performance liquid chromatography (RP-HPLC) the procedure of separating non-glycosylated proteins from glycosylated proteins can be carried out. The eluant used for the eluation of the test samples is preferably an organic solvent containing salt, such as KCl. Although various salts have been applied to the organic solvent in the prior art the effect of a Ca.sup.++ containing eluant has never been disclosed.
In the present context the term eluant is synonymous with the buffer used for eluation. The present invention provides an improved purification of the desired protein by using a Ca.sup.++ containing eluant.